P1373: UNCOVERING UPSIDES AND PITFALLS OF BASE AND PRIME EDITING IN HEMATOPOIETIC STEM CELLS
Authors: Martina Fiumara, Samuele Ferrari, Attya Omer-Javed, Stefano Beretta, Luisa Albano, Daniele Canarutto, Angelica Varesi, Chiara Gaddoni, Chiara Brombin, Federica Cugnata, Erika Zonari, Matteo Maria Naldini, Matteo Barcella, Bernhard Gentner, Ivan Merelli, Luigi Naldini
Journal: HemaSphere (2023)

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: Emerging base and prime editing (BE/PE) may provide safer and more precise genetic engineering than nuclease-based approaches bypassing the dependence on DNA double strand breaks (DSBs). Aims: However, comprehensive characterization of efficiency, tolerability and genotoxicity of these platforms is lacking and relevant to instruct safe clinical application. Methods: Here, we comparatively assessed state-of-the-art cytidine and adenine BE, such as BE4max and ABE8.20-m respectively, and PE versus Cas9 in human hematopoietic stem/progenitor cells (HSPCs) upon mRNA delivery. Results: When targeting a common locus using the same sgRNA, ABE8.20-m outperformed BE4max and Cas9 reaching up to 90% of alleles modification. Cas9-edited HSPCs displayed the lowest engraftment capacity, despite all treatments maintained multilineage reconstitution and no output skewing, as assessed by clonal tracking analyses. Cas9 editing, however, showed moderate shrinkage of clonal complexity in xenotransplanted mice. However, BE4max edited cells tended to decrease over time in the graft pointing to some detrimental response to the treatment in the long-term engrafting HSPC subset. Transcriptional analyses uncovered that BE4max trigger p53 pathway activation, albeit to lower extent as compared to Cas9 treatment, likely due to the conversion of nickase-induced DNA single-SB to DSB. Indeed, sequencing of the target locus revealed that DNA DSBs were less frequent but not abrogated by BE, particularly for the cytidine BE due to suboptimal inhibition of base excision repair. Concordantly, large deletions and translocations were detected upon BE4max treatment despite to lower frequency than upon Cas9 editing. Moreover, ultrahigh coverage whole exome sequencing on long-term xenografts uncovered a genome-wide effect of BE4max on the mutational landscape of hematopoietic clonotypes. Conversely, we did not observe detectable impact of ABE8.20-m in the same analysis. Additionally, both BEs, but not Cas9, upregulated interferon-stimulated genes, suggesting sensing of exogenous long mRNAs. mRNA engineering abolished interferon response without aggravating p53 activation and allowed >80% editing for all platforms in long-term xenografts. In addition, tailored BE4max expression resulted in more efficient BER inhibition leading to reduced proportion of DSBs and improved precision at the target site. We then applied the same mRNA engineering strategy to PE and reached >50% editing in HSPCs. Around 10% of PE alleles carried imprecise editing outcomes, including indels and long-range deletions, despite less frequently than upon Cas9 treatment. Transcriptional analyses showed upregulation of p53- and apoptosis-related genes upon PE. While some p53 targets were shared by PE and Cas9 treatments, others were specific of PE and likely due to cellular sensing of the reverse transcriptase. Despite these adverse effects reduced the hematopoietic graft size compared to mock electroporated controls, >50% PE efficiency was maintained upon serial transplantation. Summary/Conclusion: Overall, our study validated the potential for precise and effective editing of adenine BE among emerging nickase-based platforms, building confidence on its current entry into the clinical arena Keywords: CD34, HSC, Stem cell gene therapy, Genetic modifiers